Quantitative analyses sensitive to extremely low concentrations are regularly made with immunoassay techniques. The term "immunoassay" is generic to a wide variety of processes and procedures. Basically it involves a partner-specific interaction of a plurality of partner substances which are preferentially attracted to one another. A well-known example is an antibody of known specificity which is one of the partners and is to be receptive of an antigen (the other of the partners). The presence and concentration of one of them is to be detected and measured in the immunoassay procedure. As stated above, immunoassays are examples of techniques useful in other than antibody/antigen situations, as will later be discussed.
There are two basic analytical techniques, the "sandwich" technique and the "competitive" technique. Both of these techniques are widely used, and in each of them the amount of the partners bonded together is either directly measured, or is measured by way of difference. Persons skilled in this art are already fully aware of these techniques, and these techniques per se are not the instant invention.
It has long been recognized that an immunoassay is a sensitive procedure and is at least theoretically able to perform assays beyond the abilities of even the most sensitive of other known procedures. However, its processes have been very slow and inconvenient to use, sensitivity has been variable and often low, and repeatability has been well below what the process theoretically should be able to deliver. Also the consumables required for these procedures have had short shelf lives and have required more attention to their use than is desirable in what should be a highly automated and speedy procedure. This is also true of other than antibody/antigen situations.
Not surprisingly, considerable efforts have been made in the past decade to reduce surface-based analytical processes to a convenient laboratory analysis procedure. Especially strong efforts have been made in surface enhancement. Some efforts at surface enhancement have produced significant improvements, but these have been unaccompanied by suitable explanations or understandings of how these improvements occur, or for that matter, even what they are. Instead the art has developed pragmatically, step-by-step, and by now the dangers of trying definitely to explain the rationale of them have become very apparent. The newer procedures either work better, or they do not.
As it happens, tremendous increases in sensitivity can be attained with the use of any of the three best-known techniques of surface enhancement. One is surface enhanced fluorescence, another is surface enhanced Raman spectroscopy, and the other is surface enhanced colorimetric procedures. This invention improves the sensitivity, accuracy, convenience, speed and availability of all of these procedures, and provides the basis for a very effective consumable for use in these procedures.
These procedures all require the adherence of a specific partner or partners to a surface. Both planar and spherical substrates are known in the art. One means for enhancing adhesion of the partner to the substrate is to coat the surface of the substrate with a coupling agent which is adherent to the surface, and this technique is known. This invention also serves that function. Importantly, it also serves as a means accurately to space the partner from a metal-laden substrate surface, the accuracy of which spacing profoundly increases the sensitivity of the procedure, which is not, a previously known fact. Further, it also protects the partners from deleterious reactions with metals that may be placed on the substrate.
Polyglutaraldehyde ("PGA" herein) is a preferred coupling agent. A suitably prepared surface coated with PGA (or with any other suitable coupling agent) will in fact reliably retain a substantially greater amount of a partner to its surface than would be retained by a substrate surface which is not coated with such a coupling agent. This is another feature which increases the sensitivity of the procedure.
The enhanced retention of a partner by the coupling agent along with the accuracy of spacing provided by a carefully controlled thickness of coupling agent constitutes a basis for a multiplied effectiveness of the procedures.
Procedures according to this invention are a wet process. They depend on the kinetics of contact between the partner on the layer of coupling agent and the patient sample which includes the other partner. The patient sample is characteristically diluted in a buffer solution. As in all contact processes, the gross rate at which the reaction occurs is a function (among other variables) of the total surface area to be contacted, and of the transfer distance the molecules must travel or transfer to reach the PGA layer.
A method for increasing the gross rate is to increase the surface area, and to agitate the reactants to increase the likelihood of access of the partner molecules to the partner on the coupling agent layer. This is a worthwhile approach within the scope of this invention, but requires a substantial amount of buffer solution. For many immunoassay procedures this is a viable alternative. However, in many other, the cost of the buffered solution which often includes an expensive partner in wasteful amounts is extraordinarily high, and can render the procedure uneconomical. Such circumstances can exist, for example, when the buffer solution must include as a partner a clonal protein whose cost is very high.
Still, when a large increase in surface area is useful and buffer costs are affordable, a large number of small bodies such as microspheres coated with the coupling agent will advantageously be utilized. Microspheres or other very small bodies coated with a coupling agent such as PGA will retain many times the amount of partner that would be retained by even a very large number of planar surfaces such as glass slides. This is because of the very large increase in surface area of an increased number of very small bodies, especially of spheres. A principal advantage of these small substrates is that in a substantial body of liquid they enable the assay process to proceed at rates which approach those defined by solution kinetics. This contrasts with kinetics applicable for reactions at a fixed surface such as a glass plate where transfer of molecules to the PGA surface is inherently very slow.
However, and rather surprisingly, with this invention a flat plate can be utilized to provide reaction speeds approaching those of solution kinetics using microspheres, and with a large reduction in the requirement for a buffering solution. This is accomplished with the use of reaction regions of capillary dimensions, such that the partner in the buffering solution is very closely situated relative to the partner on the binding agent and are subject to capillary forces, so closely and uniformly that even without agitation the speed of the reaction approaches that which occurs in a well-stirred solution.
Another observation has been made that very small metallic metal islands (silver, for example) on a substrate to which a partner is directly adherent can result in substantial enhancement of sensitivity of the surface-enhanced analytical process. This has previously been accomplished with silver islands on flat glass plates. Here it may be observed that a smooth continuous plating of metallic silver does not produce much improvement in sensitivity, but that a toughened surface does produce at least improvement over that obtainable with the use of a smooth silver surface, although still much less than of separate islands. The reasons for all of this still reside in the realm of speculation, but it is observable that silver islands separate from one another produce a greatly improved assay, and that a suitably toughened one gives at least some improvement. As will later be disclosed, silver is only one example of a suitable metal for this purpose.
A problem solved by this invention resides in the distance-dependency of a partner from the metal islands. For fluoroscopic techniques a relatively large spacing provides best enhancement, while for Raman spectroscopy techniques, a smaller spacing provides best enhancement. In both cases there is an optimum spacing, and deviation from it results in less enhancement Accordingly, in this invention, the coupling agent has a function in addition to its capacity to bind a partner--it is to provide a very accurate spacing of its exposed surface from the substrate (including the islands) in order to improve the sensitivity of the procedure.
Whatever the situation, the art as presently known does not provide a convenient source of a known amount of partner compound for a suitably rapid analysis procedure. All surface-enhanced analytical procedures require a "consumable" which must be provided at the outset of each assay procedure. It is an object of this invention to provide a convenient consumable of known and optimized properties and good shelf life, thereby greatly improving the convenience and reliability of analyses utilizing the partners involved.
In fact, analyses using this invention are more sensitive by at least several degrees of magnitude compared to analogous analytical procedures, and complete their reactions between about 8 and 20 times faster than other known procedures. The repeatability of the procedure is greatly improved, and the analytical procedure can be simplified.
This invention provides a remarkably improved analytical procedure, and a consumable (sometimes called a "surfaced article") for accomplishing it.